Guanyong Su, Hongxia Yu,*, Michael H. W. Lam, John P. Giesy,,,, and Xiaowei Zhang*, INTRODUCTION

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1 Article pubs.acs.org/est Mechanisms of Toxicity of Hydroxylated Polybrominated Diphenyl Ethers (HO-PBDEs) Determined by Toxicogenomic Analysis with a Live Cell Array Coupled with Mutagenesis in Escherichia coli Guanyong Su, Hongxia Yu,*, Michael H. W. Lam, John P. Giesy,,,, and Xiaowei Zhang*, State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing , People s Republic of China State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, Special Administrative Region (SAR), People s Republic of China Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824, United States *S Supporting Information ABSTRACT: Results of previous studies have indicated that 6-HO-BDE-47, the addition of the hydroxyl (HO) group to the backbone of BDE-47, significantly increased the toxicity of the chemical compared to its postulated precursor analogues, BDE-47 and 6-MeO-BDE-47. However, whether such a result is conserved across polybrominated diphenyl ether (PBDE) congeners was unknown. Here, cytotoxicity of 32 PBDE analogues (17 HO-PBDEs and 15 MeO-PBDEs) was further tested and the underlying molecular mechanism was investigated. A total of 14 of the 17 HO-PBDEs inhibited growth of Escherichia coli during 4 or 24 h durations of exposure, but none of the MeO-PBDEs was cytotoxic at the concentrations tested. 6-HO-BDE-47 and 2-HO-BDE-28 were most potent with 4 h median effect concentrations (EC 50 )of and 6.25 mg/l, respectively, which trended to be lesser with a longer exposure time (24 h). Expression of 30 modulated and validated genes by 6-HO-BDE-47 in a previous study was also observed after exposure to other HO-PBDE analogues. For instance, uhpt was upregulated by 13 HO-PBDEs, and three rrna operons (rrna, rrnb, and rrnc) were downregulated by 8 HO-PBDEs. These unanimous responses suggested a potential common molecular signaling modulated by HO-PBDEs. To explore new information on mechanisms of action, this work was extended by testing the increased susceptibility of 182 mutations of transcriptional factors (TFs) and 22 mutations as genes modulated by 6-HO-BDE-47 after exposure to 6- HO-BDE-47 at the 4 h IC 50 concentration. Although a unanimous upregulation of uhpt was observed after exposure to HO- PBDEs, no significant shift in sensitivity was observed in uhpt-defective mutants. The 54 genes, selected by cut-offs of 0.35 and 0.65, were determined to be responsible for organic acid/oxoacid/carboxylic acid metabolic process pathways, which supported a previous finding. INTRODUCTION Some hydroxylated polybrominated diphenyl ethers (HO- PBDEs) have been identified as potential transformation products of polybrominated diphenyl ethers (PBDEs) and have been detected in human bodies, 1,2 which has raised concern about their potential toxicity and a need for knowledge about modes of molecular toxicity, so that accurate assessments of risk can be conducted. 3 Results of several in vivo or in vitro studies 4 6 suggested that PBDEs might undergo biotransformation. For example, biotransformation of BDE-100 to mono- HO-PBDEs was observed in both rats 5 and mice, 4 and two mono-ho-pbdes were identified in human hepatocytes exposed to BDE However, not all studies have found HO-PBDEs to be biotransformation products of PBDEs. After exposure to BDE-47 via their diet in Japanese medaka, no HO- PBDEs were detected. 7 Results of the sequential study suggested that demethylation of 6-MeO-BDE-47 was the Received: January 19, 2014 Revised: April 7, 2014 Accepted: April 9, 2014 Published: April 9, American Chemical Society 5929 dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

2 Environmental Science & Technology Article Figure 1. Structures of 17 HO-PBDE and 15 MeO-PBDE analogues. primary pathway leading to formation of 6-HO-BDE-47 in medaka, while the previously hypothesized formation of HO- PBDEs from synthetic BDE-47 did not occur. 8 Because HO- PBDEs can have a natural origin or be derived from synthetic brominated flame retardants, more attention is being paid to the HO-PBDEs, some of which are more potent for end points than the postulated precursor PBDEs and corresponding methoxylated polybrominated diphenyl ethers (MeO- PBDEs). 3 The fact that HO-PBDEs, including 6-HO-BDE-47 were detected in human blood, 1 has led to interest in the potential of HO-PBDEs to modulate gene expression and led to toxicity. Assessment of PBDEs and their analogues has mostly focused on their nuclear-hormone-receptor-mediated potency 3,9 11 or effects on reproduction 12 or neurotoxicity. 13 However, the generic toxic potency and the underlying molecular mechanisms of these brominated compounds had not been investigated previously. Genome-wide transcriptional investigations, such as whole cell arrays, 14,15 are high-throughput methods to determine toxicological mechanisms of target chemicals and could serve as a tool for a number of reverse genetic approaches. Unlike microarray technology, the live cell array can avoid complex protocols of pretreatment, high-cost experimental materials, interferences, and lack of temporal resolution but can achieve comparable results, such as toxicity identification and pollutantspecific molecular fingerprints. 16 From the use of genome-wide 5930 dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

3 Environmental Science & Technology Article Figure 2. Quantitative expressions of 30 genes in E. coli after a 4 h exposure to 17 HO-PBDE analogues. The fold change of gene expression is indicated by the color gradient on the right. The chemical and gene identifiers can be found on the right and bottom of the plot, respectively. live cell arrays, cytotoxicity of 6-HO-BDE-47 and its two analogues, BDE-47 and 6-MeO-BDE-47, and the associated molecular mechanisms had already been assessed in previous studies, which indicated that potency of 6-HO-BDE-47 to inhibit growth of Escherichia coli was greater than its postulated precursor BDE-47 and corresponding 6-MeO-BDE-47 and identified potential pathways and transcriptional networks modulated by 6-HO-BDE However, whether such a mechanism is conserved across PBDE congeners was unknown. Recently, the gene loss-of-function screen assay provides another new resource for not only systematic analyses of unknown gene functions and gene regulatory networks but also genome-wide testing of mutational effects in E. coli K Changes in sensitivities of mutants offers another way of further characterizing mechanisms of action and might be a good addition to newly developed high-throughput studies. 18 In previous studies, 15 the addition of the hydroxyl (HO) group to the backbone of BDE-47 normally caused stronger potency that that of the methoxy (MeO) functional group. To investigate whether this effect occurs among various PBDE congeners, cytotoxicity of 32 PBDE analogues (17 HO-PBDEs and 15 MeO-PBDEs) was tested. The results of those studies suggested that only HO-PBDEs can cause toxicity to E. coli at concentrations that do not exceed solubility. To explore possible common molecular signaling among different HO- PBDE congeners, 30 genes modulated by 6-HO-BDE-47 were also assessed for transcriptional responses of E. coli to 16 other HO-PBDEs. Then, using 6-HO-BDE-47 as a model chemical, 182 gene knockout mutants, including all transcriptional factors (TFs) and modulated genes by 6-HO-BDE-47, were assessed to determine shifts in sensitivity after exposure to 6-HO-BDE- 47 at the IC 50. On the basis of shifts in patterns of sensitivity, an assessment of transcriptional networks, including all TFs, was conducted to determine possible mechanisms of toxicity of 6- HO-BDE dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

4 Environmental Science & Technology Article Figure 3. Real-time, quantitative expression of uhpt in E. coli after exposure to various HO-PBDE analogues. Exposure to lesser, moderate, and greater concentrations of target compounds was represented by the lower, middle, and upper bands in each gene column. The fold change of gene expression is indicated by the color gradient on the right, and the time course of expression changes is indicated from left to right. MATERIALS AND METHODS Chemicals and Reagents. Analogues of PBDEs, including 17 HO-PBDEs (Figure 1) and 15 MeO-PBDEs (see Figure S1 of the Supporting Information), were synthesized in the Department of Biology and Chemistry of City University of Hong Kong following previously published methods. 19 Purities of the synthesized compounds were determined to be greater than 98%. The results of proton nuclear magnetic resonance ( 1 H NMR) and electrospray liquid chromatography tandem mass spectrometry (LC MS/MS) were used to determine that unwanted intermediates and end products were not produced during the synthesis. In particular, there were no detectible polybrominated dibenzo-p-dioxins/furans. 8 Live Cell Array and E. coli Keio Collection. The microbial live cell array collection, including more than 1800 of 2500 promoters in the entire genome of E. coli K-12 strain MG1655, was used to assess differential expression or genes by use of previously described methods. 15 Each of the reporter strains was coupled with a bright, fast-folding green fluorescent protein (GFP) fused to a full-length copy of an E. coli promoter in a low-copy plasmid. This enabled measurement of expression of genes within minutes with high accuracy and reproducibility. The tested knockout clones (strain background: E. coli K-12 BW25113) carried a deletion of a single gene, with a kanamycin resistance gene serving as the replacement, which were developed as a collaboration between the Institute for Advanced Biosciences, Keio University (Tokyo, Japan), Nara Institute of Science and Technology (Ikoma, Japan), and Purdue University (West Lafayette, IN). All clones from either live cell array or gene knockout collections were grown at 37 C in lysogeny broth (LB) Lennox media plus 25 mg/l kanamycin. Cytotoxicity. Stock solutions of test chemicals (2000 mg/ ml) were prepared in dimethyl sulfoxide (DMSO, Tedia, Fairfield, OH), and other stock solutions were made by serial dilution with DMSO. Eight different concentrations of HO- PBDE analogues (100, 25, 6.4, 1.6, 0.39, 0.098, 0.024, or mg/l) (n = 3) were used in the E. coli cytotoxicity test. After 4 or 24 h of incubation at 37 C, growth of E. coli was determined by measurement of optical density (OD) at 600 nm, by use of a Synergy H4 hybrid microplate reader (BioTek Instruments, Inc., Winooski, VT). In parallel, 10 μl of Alamar blue (Beijing CellChip Biotechnology, Inc., Beijing, China) was added to 150 μl of LB medium for each well to assess cell viability after 3 h of incubation, according to the ratio. Alamar blue was known to be nontoxic to cells. After cells were stained with Alamar blue for 1 h, blue red fluorescence was detected by a Synergy H4 hybrid microplate reader (excitation/emission, 545/590 nm) (BioTek Instruments, Inc., Winooski, VT). Expression of Genes. Exposure was performed with a slight modification of previously described methods. 20 Strains of E. coli were inoculated into a fresh 96-well plate from a 96- well stock plate by use of disposable replicators (Genetix, San Jose, CA). Cells were incubated at 37 C for 3.0 h in a 96-well plate and then transferred into a 384-well plate. Finally, 3.79 μl of DMSO (solvent control) or chemical stock solutions were added into individual wells on the 384-well plate to make a final concentration of 0, 1, 10, and 100 mg of chemical/l, except for 2 -HO-BDE-28 and 6-HO-BDE-47. These two chemicals, 2 - HO-BDE-28 and 6-HO-BDE-47, killed E. coli at a concentration of 100 mg/l, and their exposure concentration were 5932 dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

5 Environmental Science & Technology Article Figure 4. Shifts in sensitivity of TF-defective mutants by exposure to 6-HO-BDE-47 and their regulated network in E. coli. Each gene is displayed by a circular node, and the transcriptional interaction between TF TF is indicated by an arrow edge. Different colors in the node represent the E. coli growth against the 4 h IC50 concentration of 6-HO-BDE-47. Green nodes represented resistant strains using a cutoff of 0.65; blue nodes represented sensitive strains using a cutoff of 0.35; and the white nodes represented the strains with no significant sensitivity changes. The pink nodes represent genes that are absent in the E. coli Keio collection. made up to be 0, 0.01 EC20, 0.1 EC20, and EC20, respectively. GFP intensity of each well was consecutively monitored every 10 min for 4 h by a Synergy H4 hybrid microplate reader (excitation/emission, 485/528 nm) (BioTek Instruments, Inc., Winooski, VT). Assessment of Shifts in Sensitivities. To characterize the sensitivity of specific gene knockout strains, clones of interest were screened by comparing to the 4 h IC50 concentration of 6HO-BDE-47 obtained in the initial wide-type screen in two rounds. After thawing, clones of interest were inoculated from the stock plates into a 15 ml centrifuge tube with fresh medium and pre-cultured overnight for the sensitivity screening. In each clone of each round, there was also a control group in two replicates without target chemical addition to ensure its robust growth. All conditions were duplicated. Plates were incubated at 37 C for a 3 or 23 h period for a total of 4 or 24 h exposure, and then 4 μl of Alamar blue was added to 75 μl of LB medium for each well to assess cell viability. After 1 h of incubation with Alamar blue, the blue red fluorescence was detected by a Synergy H4 hybrid microplate reader (excitation/ emission, 545/590 nm). Statistical Analysis and Data Visualization. Structures of HO-PBDE analogues were generated with ChemBioDraw Ultra 11.0 (Figure 1). A gene expression heat map of uhpt was conducted by use of R software (Figure 2). For temporal gene expression, a linear regression model was applied to select promoter reporters, of which expression was significantly differentiated relative to exposure to the chemicals (Figure 3). Genes were considered to be altered significantly only when their p values were less than The TF TF interactions were looked up in a RegulonDB database, and the visualized transcriptional network was conducted by use of Cytoscape, version (Figure 4).21 Gene set enrichment analysis (GSEA) was performed on R version using the GOstats package, with R script tailored to E. coli dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

6 Environmental Science & Technology Article Table 1. Cytotoxicity End Points of 17 HO-PBDEs after a 4 and 24 h Exposure to E. coli a compounds 24 h EC b 50 (mg/l) 4 h EC 50 (mg/l) 24 h LOEC c (mg/l) 4 h LOEC (mg/l) 24 h MII d (%) 4 h MII (%) 4-HO-BDE-90 NA e NA 100 NA 13 NA 3-HO-BDE-100 NA NA 100 NA 13 NA 2 -HO-BDE-66 NA NA HO-BDE-68 NA NA 25 NA 24 NA 6-HO-BDE-85 NA NA HO-BDE-123 NA NA 6.25 NA 27 NA 5-Cl-6-HO-BDE-47 NA NA 25 NA 29 NA 6-HO-BDE-90 NA NA 6.25 NA 31 NA 6 -Cl-2 -HO-BDE-68 NA NA 25 NA 32 NA 2 -HO-BDE HO-BDE HO-BDE-17 NA NA NA 100 NA HO-BDE-17 NA NA NA 25 NA HO-BDE-7 NA NA NA 100 NA HO-BDE-7 NA NA NA NA NA NA 4 -HO-BDE-49 NA NA NA NA NA NA 2 -HO-BDE-25 NA NA NA NA NA NA a Vaules are shown as the mean of three replicates. EC 50 means median effect concentration. LOEC means lowest observed effect concentration. MII means maximum inhibition induction. e NA means not achieved. RESULTS AND DISCUSSION that E. coli might undergo several similar molecular pathways Cytotoxicity. After a 4 or 24 h exposure, no inhibition of while exposed to different HO-PBDE analogues. After a 4 h cell division was observed for 15 MeO-PBDEs at concentrations ranging from 0 to 25 mg/l. However, cell division of E. exposure, fold changes of 30 genes were reported in a heat map (Figure 2), which showed that exposure to HO-PBDEs results coli cells was significantly inhibited after exposure to 14 of 17 in fewer upregulated gene reporter stains than downregulated HO-PBDEs, especially for 6-HO-BDE-47 and 2 -HO-BDE-28, strains. The gene uhpt was classified into one group from the which showed a concentration-dependent effect including total other 29 genes and mainly showed an upregulated profile. inhibition of growth of bacterial at 100 mg/l (Table 1). This Three rrna operons (rrna, rrnb, and rrnc) were finding indicated that HO-PBDEs were more cytotoxic to E. consistently downregulated following exposure to HOcoli cells than MeO-PBDEs. The more cytotoxic potency HO- PBDEs, while uhpt was upregulated by 14 HO-PBDE PBDEs was also reported in previous publications involving analogues (Figure 3 and see Figure S3 of the Supporting H295R cells 10 or zebrafish embryos. 22 Differences between the Information). In E. coli, the uhpt gene, a P i -linked hexose two functional groups, HO or MeO, were presumed to be the phosphate antiport carrier, 26 is a member of the major main reason for the observed differential toxic potencies. facilitator superfamily, which can catalyze active transport of Hydroxyl groups can form hydrogen bonds with water sugar phosphates by an obligatory exchange mechanism. molecules to enhance water solubility and also play an Expression of uhpt is controlled by a two-component important role in enzymatic reactions. Among the 17 tested regulatory system consisting of histidine kinase uhpb and the HO-PBDEs, 6-HO-BDE-47 and 2 -HO-BDE-28 were most response regulator uhpa, 27 which are normally involved in cytotoxic, with median effect concentrations (EC 50 ) of responses of bacteria to environmental stimuli. 28 Fosfomycin is and 6.25 mg/l with a 4 h exposure, respectively. When the known to be transported into cells via the glpt and uhpt duration of exposure was 24 h, the EC 50 values of 6-HO-BDE- transporters, 29 which indicated that disturbance of glucose-6-47 and 2 -HO-BDE-28 were 9.68 and 4.28 mg/l, respectively, phosphate acquisition might be a common signaling response which indicated that longer term exposure to HO-PBDEs during exposure to HO-PBDEs. In mammals, the alignment of resulted in greater toxicity to E. coli. Both 6-HO-BDE-47 and the sequence of amino acids suggested that uhpt belongs to a 2 -HO-BDE-28 have been shown to be produced naturally, family of transporters of phosphorylated metabolites, including 2,23,24 and humans might be exposed to these chemicals via consumption of sea food. 25 Profiles of Gene Expressions. Expression of 30 genes that had been shown previously to be modulated by 6-HO-BDE-47 in the microbial reporter stains was also observed after exposure to 16 other HO-PBDE analogues and exhibited both time- and concentration-dependent responses (see Figure S4 of the Supporting Information). Each specific HO-PBDE analogue induced a different pattern of expression of genes, which suggested complex mechanisms of effects of HO-PBDEs on toxicogenomic responses. However, common molecular signals were also observed in profiles of expression of genes. A total of 8 of 30 genes, evga, hupb, rrnb, flgm, rrna, rrnc, seru, and uhpt, can be modulated by more than 10 HO-PBDE analogues the glucose-6-phosphate transporter, 30 which also posed an indication related to membrane transporter disease after exposure to HO-PBDEs in mammals. The three rrna operons (rrna, rrnb, and rrnc) belong to 16S rrna, which is a component of the 30S small subunit of prokaryotic ribosomes. Because of its high conservation between different species of bacteria and archaea, these genes were usually used for phylogenetic studies. 31 The 16S rrnas also play an important role in subunit association and translational accuracy in bacteria. Downregulation of the rrna operons by HO- PBDEs might be another common signaling adaptive response. Shifts in Sensitivities of Mutants. The gene loss-offunction screen assay provides another new method for genome-wide testing of mutational effects for specific cytotoxic (see Figure S2 of the Supporting Information), which indicated contaminants, 17 such as zinc-containing nanoparticles dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

7 Environmental Science & Technology Considering that 6-HO-BDE-47 was strongly cytotoxic to E. coli, a mutant library, including 182 mutations in TFs and 22 mutations as genes modulated by 6-HO-BDE-47, was constructed to assess shifts in their sensitivities after exposure to 6-HO-BDE-47 at the IC 50 for 4 h. Some genes that are essential for growth of bacteria are not available in the E. coli Keio collection. For each mutated strain, cytotoxicity was determined in two cycles. Information on inhibition collected in two rounds was fitted to a linear function (y = x) with adjusted R 2 values of or after 4 or 24 h exposure, respectively, which ensured its robustness (see Figure S5 of the Supporting Information). Differences in cytotoxicity between 4 and 24 h exposure groups were also compared via their 95% confidence ellipses of mean or prediction over the collected data points (see Figure S5 of the Supporting Information). Responses observed after a 24 h exposure tended to be less than those after a 4 h exposure, which indicated that the mutants would exhibit stronger inhibition after a longer duration of exposure. Great concerns existed on the relationships among different high-throughput screening technologies. For example, correlations between sensitivity profiles and results of studies with microarrays have been proposed previously, but few correlations were observed between profiles of genes during global genome monitoring and sensitivity of mutants of E. coli. 17,32 To explore potential relationships between expression genes and profiles of sensitivities of mutants, 22 selected genes that were modulated by the model chemical 6-HO-BDE-47 were also assessed to determine sensitivities of mutant cell lines after prior exposure to the IC 50 concentration of 6-HO-BDE-47 for 4 h. Here, growth of 22 mutation strains, expressed as the ratio between the exposed and control, ranged from 0.34 ± 0.03 to 0.69 ± Although expression of uhpt can be modulated by most of the tested HO-PBDE analogues, growth of its mutation strain was not significantly different from growth of the widetype strain. After exposure to 6-HO-BDE-47, no clear evidence of a relationship between expressions of genes and shifts in sensitivity of growth of mutation strains was observed in this research. A mutation library consisting of a whole transcriptional network in E. coli was also introduced during the study, the results of which are presented here to study changes in sensitivity to exposure to 6-HO-BDE-47 against the 4 h IC 50 concentration (Figure 4). In molecular biology and genetics, a TF is usually regarded as one of the most common mechanisms used by cells to control when genes are switched on or off. 33 TFs are also known as one of the groups of proteins that read and interpret the genetic blueprint in DNA, by binding to DNA and helping to initiate a program of increased or decreased gene transcription. RegulonDB, the primary reference database of the best-known regulatory network of any free-living organisms, has summarized 202 TFs and their associated TF TF transcriptional relationships (see Table S1 of the Supporting Information), which make it easier to determine how a particular TF interacts with multiple other TFs while in up- or downregulated situations. 34 On the basis of their observed interactions among all TFs, changes in sensitivity were observed over the entire transcriptional network. Based on growth cutoffs of 0.35 and 0.65, 8 and 46 strains were selected as those carrying the most sensitive and resistant genes to 6- HO-BDE-47. Because of their important roles in development, intercellular signaling, and cell cycle, mutations in TFs are associated with specific diseases in organisms. 35 Here, the 54 Article mutant strains affected by 6-HO-BDE-47 were also taken as selected genes for a hypergeometric-based test, and this gene ontology (GO) term association analysis strongly suggested that 6-HO-BDE-47 caused toxicity to bacteria through an organic acid metabolic process, an oxoacid metabolic process, and a carboxylic acid metabolic process (p < 0.05; see Table S2 of the Supporting Information). HO-PBDEs contain the phenol group, which can confer weak acidity and be generally referred to as organic acids. This finding supported a previous report that 6-HO-BDE-47 was difficult to be transformed into other corresponding products during in vitro microsomal exposure studies. 7 Implications. PBDEs and their metabolites (HO-PBDEs and MeO-PBDEs) are of interest because of their potential effects on people, especially HO-PBDEs, which showed a more potent receptor activity compared to those of the parent PBDEs and corresponding MeO-PBDEs. 3,15,25 Previous studies showed that HO-PBDEs were detected even in human blood, 1 which indicated that formation of HO-PBDEs from PBDEs might occur via endogenous transformation. Studies also showed that HO-PBDEs can be formed from naturally occurring MeO-PBDEs 7,8,23 and can be detected in various marine organisms, 25,39 which also posed another route of dietary exposure to HO-PBDEs by humans, this observation resulted in considerable concern about the toxic mechanism of these accumulative transformation products of naturally occurring and synthetic PBDEs. In our study, HO-PBDEs inhibited growth of E. coli rather than MeO-PBDEs, which ranked HO-PBDEs to an even greater risk. We also found that several similar molecular signalings would be modulated after exposure to different HO-PBDEs. For example, uhpt can be upregulated by 13 of the 17 HO-PBDEs, and three rrna operons (rrna, rrnb, and rrnc) can be downregulated by 8 HO- PBDEs, which proposed possible mechanism of toxicity of HO- PBDEs. Further work should be conducted to uncover connections between the molecular responses in prokaryotic and eukaryotic cells and to investigate the toxicity potencies of HO-PBDEs in human cells. ASSOCIATED CONTENT *S Supporting Information Transcriptional relationships between TFs summarized by RegulonDB (RegulonDB, version 8.0), also known as the primary reference database of the best-known regulatory network of any free-living organisms (Table S1), gene set enrichment analysis on 54 sensitive-shift genes to 6-HO-BDE- 47 against the 182 TF mutations in the Keio collection (Table S2), structures of 15 MeO-PBDE analogues (Figure S1), altered genes by different HO-PBDEs (Figure S2), real-time, quantitative expression of rrna/b/c/h in E. coli after exposure to various HO-PBDE analogues (Figure S3), real-time, quantitative determination of mrna abundances as measures of 30 selected genes in E. coli after exposure to 17 HO-PBDE analogues (Figure S4), and linear fitting of data points collected in two cycles to demonstrate shifts in sensitivities of mutants pre-exposed for 4 h to the IC 50 concentration of 6-HO-BDE-47 (Figure S5). This material is available free of charge via the Internet at AUTHOR INFORMATION Corresponding Authors *Telephone: Fax: yuhx@nju.edu.cn dx.doi.org/ /es Environ. Sci. Technol. 2014, 48,

8 Environmental Science & Technology *Telephone: Fax: Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The research was supported by the National Natural Science Foundation of China (Grant ) and the National High-Tech Research and Development Program of China (863 Program, Grant 2013AA06A309). This project was also supported by the Jiangsu Provincial Key Technology Research and Development Program (BE ) and the Collaborative Innovation Center for Regional Environmental Quality. John P. Giesy was supported by the program of 2012 High Level Foreign Experts (GDW ) funded by the State Administration of Foreign Experts Affairs, the People s Republic of China to Nanjing University, and the Einstein Professor Program of the Chinese Academy of Sciences. John P. Giesy was also supported by the Canada Research Chair Program and a Visiting Distinguished Professorship in the State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong. REFERENCES (1) Qiu, X. H.; Bigsby, R. M.; Hites, R. A. Hydroxylated metabolites of polybrominated diphenyl ethers in human blood samples from the United States. Environ. Health Perspect. 2009, 117 (1), (2) Athanasiadou, M.; Cuadra, S. N.; Marsh, G.; Bergman, A.; Jakobsson, K. Polybrominated diphenyl ethers (PBDEs) and bioaccumulative hydroxylated PBDE metabolites in young humans from Managua, Nicaragua. Environ. Health Perspect. 2008, 116 (3), (3) Kojima, H.; Takeuchi, S.; Uramaru, N.; Sugihara, K.; Yoshida, T.; Kitamura, S. Nuclear hormone receptor activity of polybrominated diphenyl ethers and their hydroxylated and methoxylated metabolites in transactivation assays using Chinese hamster ovary cells. Environ. Health Perspect. 2009, 117 (8), (4) Staskal, D. 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10 1 2 Mechanisms of Toxicity of HO-PBDEs Determined by Toxicogenomic Analysis with Live Cell Array Coupled with Mutagenesis in E. coli Guanyong Su 1, Hongxia Yu 1,*, Michael H. W. Lam 3, John P. Giesy 1,2,3,4, Xiaowei Zhang 1,* 1 State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China 2 Department of Biomedical Veterinary Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada 3 Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong, SAR, China 4 Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA Authors for correspondence: School of the Environment Nanjing University Nanjing, , China Tel: Fax: yuhx@nju.edu.cn (Hongxia Yu) howard @yahoo.com (Xiaowei Zhang)

11 Table S1. Transcriptional relationships between transcriptional factors summarized by RegulonDB (RegulonDB v8.0), also known as the primary reference database of the best-known regulatory network of any free-living organisms (In the following table, TF 1 and TF 2 represent transcription factor names; Re means regulated (+activator, -repressor, +-dual,?unknown). 1 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 TF 1 Re TF 2 acrr - acrr crp + glcc csgd? csgd fis + hns h-ns - appy lexa - yafq norr - norr rcsab + rcsa ada + ada crp + glng cspa + hns fis + hupa h-ns - bola lldr +- lldr nsrr - dsdc rcsb - gadx ada - ada crp - glng cusr + cusr fis - hupb h-ns - cadc lldr - lldr nsrr - fear rcsb + ydeo ada + aidb crp + glpr cynr - cynr fis + mara h-ns - caif lrha - flhc nsrr - fhla relb - relb adiy + gadx crp + gutm cysb + cbl fis + marr h-ns - evga lrha - flhd nsrr - fliz relb - rele agar - agar crp + hupa cysb - cysb fis + maze h-ns + flhc lrha + lrha nsrr - ihfa relb-rel - relb aidb - aidb crp + hupb cytr - cytr fis - mtlr h-ns + flhd lrp - aidb nsrr - lrp relb-rel - rele alas - alas crp + hyfr dan + ttdr fis - xylr h-ns + fliz lrp - lrp nsrr - norr rhar + rhar allr - alls crp + idnr dcur + dpia flhdc + fliz h-ns - gade lrp + malt ntrc + cbl rhar + rhas alsr - rpir crp + laci dhar - dhar fliz - csgd h-ns - gadw lrp + stpa ntrc + glng rhas + rhar arac + arac crp + lsrr dinj - dinj fliz - flhc h-ns - gadx lrp + yeil ntrc - glng rhas? rhar arac +- arac crp + mali dinj - yafq fliz - flhd h-ns + gutm lsrr - lsrr ntrc + nac rhas + rhas arac - arac crp - mali dnaa +- dnaa fliz - gade h-ns - hdfr lysr - lysr ompr - bola rhas? rhas arca - beti crp + malt dnaa - dnaa fliz - mlra h-ns - hns mali - mali ompr + csgd rob + mara arca + dpia crp + mara dpia - appy fucr + fucr h-ns - leuo mara + mara ompr - flhc rob + marr arca - fnr crp + marr dpia + dpia fur - flhc h-ns - lrp mara + marr ompr - flhd rob - rob arca + gadx crp + maze dsdc - dsdc fur - flhd h-ns + maze mara + puta oxyr + fur rsta - csgd arca - lldr crp + melr evga + evga fur - fnr h-ns - nhar mara - rob oxyr - oxyr rutr - gadw arca - puur crp + mhpr evga + gade fur - fur h-ns - rcsa marr - mara pdhr - pdhr rutr - gadx arca + xylr crp + mlc evga + ydeo fur - metj h-ns - rcsb marr - marr pepa - pepa rutr - nemr argp - argp crp - mlc exur - exur fur - nac h-ns + srlr mata + ecpr phob + argp rutr - rutr argp + dnaa crp + mtlr exur - uxur fur - purr h-ns - stpa mata - flhc phob - cra sdia + gadw argr - argr crp +- nagc fnr + arca fur - soxr h-ns - ydeo mata - flhd phob + cusr sgrr - sgrr arsr - arsr crp + ompr fnr - arca fur - soxs hcar - hcar mata - fliz phob - fear soxr - soxr ascg - prpr crp - ompr fnr + cadc gade + gade hdfr - flhc maze - maze phob + gadw soxr + soxs asnc - asnc crp + oxyr fnr + caif gade + gadw hdfr - flhd melr + melr phob + gadx soxs + fur baer + baer crp + pdhr fnr + dcur gade + gadx hipab - hipb melr - melr phob + phob soxs + mara basr + csgd crp + prpr fnr - dpia gade + lrp hipb - hipb metj - metr phob - prpr soxs + marr basr - puta crp + rbsr fnr + fhla gade + rcsa hyfr + hyfr metr - metr phob + ydfh soxs - rob beti - beti crp + rhar fnr - fnr gade-rcs + gadx ihf + csgd mlc - malt phop + cysb soxs - soxs bglj + leuo crp + rhas fnr - gadw gadw + gade ihf + fhla mlc - mlc phop + gade stpa - leuo crp + arac crp + srlr fnr - gadx gadw - gadw ihf + fis mlra + cadc phop + gadw stpa - stpa crp + caif crp + tdca fnr - hyfr gadw +- gadx ihf - flhc mlra + csgd phop + phop tdca + tdca crp + chbr crp + uxur fnr - nac gadw - gadx ihf - flhd mngr - mngr phop - phop tdcr + tdca crp - comr crp + xylr fnr - narl gadx + gade ihf + fnr mode + narl phop + rsta torr - gadx crp + crp crp - yiaj fnr + nikr gadx + gadw ihf + hipb mpra - mpra phop - trer torr - torr crp - crp crp + zrar fnr +- pdhr gadx - gadw ihf - ihfa mtlr - mtlr phop - ydeo trpr - trpr crp + csgd cadc + cadc fnr - soxr gadx + gadx ihf - ihfb murr +- murr prpr - prpr tyrr - tyrr crp +- cytr cdar + cdar fnr - soxs gadx + hns ihf - ompr nac - asnc pspf - pspf uidr - uidr crp + dcur chbr +- chbr fnr + tdca galr - galr ihf + tdca nac - nac purr - purr uxur - uxur crp - dpia cpxr + baer fnr - yeil galr - gals ihf + yeil nagc - chbr puta - puta xylr + xylr crp + fear cpxr + cpxr fnr + yqji gals - galr ihf - yiaj nagc - creb puur - puur ydeo + gade crp + fis cpxr - csgd fadr + fadr gals - gals iclr - iclr nagc - nagc qseb + flhc ydeo - ydeo crp +- fis cra + beti fadr + iclr gcva - gcva idnr + idnr narl - caif qseb + flhd yefm - yefm crp + flhc cra + csgd fhla + fhla glcc - glcc ilvy - ilvy narl - dcur qseb + qseb yeil + yeil crp + flhd cra - glcc fhla + hyfr gntr - idnr iscr - iscr narl - dpia rbsr - rbsr yiaj - yiaj crp + fucr cra - mara fis - crp gutm + gutm leuo - cadc narl - nikr rcda + csgd yqji - yqji crp + fur cra - marr fis - fis gutm + srlr leuo + leuo nemr - nemr rcnr - rcnr zrar + zrar crp - gade cra - mtlr fis - gadx gutr - gutm leuo +- leuo nhar + nhar rcsab - csgd crp - gadx cra - pdhr fis - glcc gutr - srlr lexa - dinj nhar? nhar rcsab - flhc crp + gals csgd + csgd fis + glng h-ns - adiy lexa - lexa nikr - nikr rcsab - flhd

12 Table S2. Gene Set Enrichment Analysis on 54 sensitive-shift genes to 6-HO-BDE-47 against the 182 TF mutations in Keio collection. (The association of gene ontology was performed by R version using GOstats package. The universe and selected genes were defined according 182 mutations in the Keio collection and 54 selected sensitive or resistant genes, respectively. The analysis was conducted basing on the biological processing (BP) GO ontology. The revised R script, which was tailored according to E. coli strain MG1655, is available upon request.) GOBPID P-value OddsRatio ExpCount Count Size Term 1 GO: organic acid metabolic process 2 GO: oxoacid metabolic process 3 GO: carboxylic acid metabolic process 4 GO: small molecule metabolic process 5 GO: Inf polysaccharide metabolic process 6 GO: Inf amine biosynthetic process 7 GO: Inf cellular amine metabolic process 8 GO: Inf cellular biogenic amine biosynthetic process 9 GO: Inf carbohydrate biosynthetic process 10 GO: Inf polysaccharide biosynthetic process 11 GO: Inf cellular biogenic amine metabolic process 12 GO: amine metabolic process 13 GO: cellular amino acid metabolic process

13 45 Figure S1 Structures of 15 MeO-PBDEs analogues

14 48 Figure S2 Altered genes by different HO-PBDEs (sky blue: not altered; light pink: altered) 49 50

15 Figure S3. Real-time, quantitative expression of rrna/b/c/h in E. coli after exposure to various HO-PBDEs analogues. Exposure to lesser, moderate and greater concentration of target compounds were represented by the lower, middle and upper bands in each gene column. The fold change of gene expression is indicated by color gradient on the right, and the time course of expression changes is indicated from left to right

16 Figure S4. Real-time, quantitative determination of mrna abundances as measures of 30 selected genes in E. coli after exposure to 17 HO-PBDEs analogues. Exposures to low, medium and high concentration of HO-PBDE were represented by the lower, middle and upper bands in each gene column. Classification and visualization of the gene expression were derived by use of ToxClust 2. The dissimilarity between genes was calculated by the Manhattan distance between the gene expressions at all the concentration vs. time combinations. The fold change of gene expression is indicated by color gradient, and the time course of expression changes is indicated from left to right HO-BDE-90

17 HO-BDE-85

18 Cl-6-HO-BDE-47

19 Cl-2 -HO-BDE-68

20 HO-BDE-49

21 HO-BDE-47

22 HO-BDE-68

23 HO-BDE-17

24 HO-BDE-17

25 HO-BDE-28

26 HO-BDE-7

27 HO-BDE-7

28 HO-BDE-25

29 HO-BDE-66

30 HO-BDE-100

31 HO-BDE-90

32 HO-BDE-123

33 Figure S5 Linear fitting of data points collected in two cycles to demonstrate shifts in sensitivities of mutants pre-exposed for 4 h to IC50 concentration of 6-HO-BDE-47. The upper one represented a short-term exposure experiment, and the lower one represented a long-term exposure experiment. The red line displayed a fitted line, and the pink ellipse enclosed 95% of data points

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